Nitric Oxide Signaling in Oxytocin-Mediated Cardiomyogenesis - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 389570\|回复: 253	go Nitric Oxide Signaling in Oxytocin-Mediated Cardiomyogenesis [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 00:57 |只看该作者 |倒序浏览 |打印

作者：Bogdan A. Danalachea,b, Joanne Paquina, Wang Donghaob, Ryszard Grygorczykb, Jennifer C. Moorec, Christine L. Mummeryc, Jolanta Gutkowskab, Marek Jankowskib作者单位：aLaboratoire de Neuroendocrinologie Dveloppementale, Dpartement de Chimie et de Biochimie, Universit du Qubec  Montral, Montral, Qubec, Canada;bCentre de Recherche, Centre Hospitalier de l




         * |4 ?/ w; V) K, e+ Q, [( }

         9 ?7 i+ r% s- H$ Z+ L

                  + c, w1 A, j' o
      0 P+ G; f/ u5 G' H, [' {5 N2 }- a
      7 M' F, F; K5 D! `; y
      1 x: J2 e2 l* v& l
      【摘要】
   Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain-2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO-sensitive guanylyl cyclases. The NO generator S-nitroso-N-acetylpenicillamine (SNAP) reversed the L-NAME inhibition of cell beating and GFP expression. In OT-induced cells, L-NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, -myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT-induced P19Cl6-GFP cells revealed ventricular CM coexpressing sarcomeric -actinin and GFP, with some cells exclusively expressing -actinin, most likely of the SM phenotype. The OT-mediated production of CM, but not SM, was diminished by L-NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of L-NAME. Surprisingly, L-NAME alone decreased the expression of anti-stage specific embryonic antigen-1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6-GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.
      【关键词】 Embryonal carcinoma Oxytocin Nitric oxide Fluorescent protein reporter genes In vitro differentiation Myogenesis
   INTRODUCTION* K- _  }' a" w# b: g
, N4 d5 \4 A' z( W0 g
The in vitro differentiation of embryonic stem cells into cardiomyocytes (CM) is a poorly defined, inefficient, and relatively nonselective process . Identifying and optimizing these conditions are important as new approaches to probe the molecular mechanisms of CM differentiation and apply this knowledge to producing CM for cardiac therapy.
+ J+ \0 e1 f/ V# s, e: ]% p5 X
Oxytocin (OT), recently recognized as a cardiac hormone . We hypothesize that OTR signaling is linked to NO in the process of differentiation of stem cells into CM. Experiments were performed on pluripotent P19EC cells and, in addition, on the P19Cl6-GFP cell line to investigate differentiation in response to OT treatment.
% U) T+ K9 P, |' S
MATERIALS AND METHODS9 E. x. c2 |8 z# x

Cell Culture and Differentiation) A9 s& y1 h2 P" k9 i' D: n

P19EC cells were propagated and differentiated as described .
/ a. G& S* ?& @- N9 Y
Microscopic Analysis/ L# _+ r+ |/ \7 ?) x/ a

Cellular morphology, MLC-2v-GFP fluorescence and some protein markers were examined with an inverted microscope (Carl Zeiss, Jena, Germany http://www.zeiss.com) equipped for epifluorescence analysis. Micrographs were taken with a Nikon Coolpix 5,000 camera (Nikon, Tokyo, Japan, http://www.nikon.com) and fluorescent areas analyzed with Image J software (National Institutes of Health, Bethesda, MD, http://www.nih.gov) employing the threshold function. Immunocytochemistry was performed as described elsewhere . The mouse monoclonal antibody immunoglobulin M (IgM) anti-stage specific embryonic antigen-1 (SSEA-1) was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health & Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). Goat polyclonal antibody IgG anti-sarcomeric myosin heavy chain (MHC; antibody K-16) and IgG anti-dihydropyridine receptor- 1 (DHPR; antibody N-19) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, http://www.scbt.com/). Secondary antibodies were conjugated to fluorescein (Santa Cruz Biotechnology).+ q7 K, \# m4 q4 D

Confocal Microscopy Analysis5 T8 B( d; o$ M. E

P19Cl6-GFP cells, subjected to different treatments and collected on day 14, were stained for -actinin with indirect Alexa Fluor 568 antibody complex. Anti-sarcomeric -actinin monoclonal antibody, which reacts with skeletal and cardiac muscle -actinins (clone EA-53 from Sigma Chemical Co.) was used at 1:800 dilution. Goat antimouse IgG secondary antibody conjugated to red fluorophore Alexa Fluor 568 was from Invitrogen Life Technologies (Burlington, Ontario, Canada, http://www.invitrogen.com). Image analysis was performed in MRC1024 confocal microscope (Bio-Rad Microscience, Cambridge, MA, http://www.bio-rad.com) equipped with a Krypton argon laser (excitation at 488 nm, 568 nm) combined with an Eclipse Model TE 3000 inverted microscope (Nikon) with emission filters measuring green and red fluorescence at wavelengths of 488 and 568 nm. The image was registered by LaserSharp V3.2 software (Bio-Rad). The Image J program and intensity correlation analysis software were deployed to calculate green fluorescence, red fluorescence, and their colocalization areas, and expressed as a percentage of the total image area.) C, j. o# h( e# f, ]& ~+ Y$ J
% D/ D. ^8 U5 U' m# @
OT Radioimmunoassay1 Q' U# e; U' v/ W7 S7 R/ a% w

P19EC cultures induced by OT or DMSO were incubated with fresh -MEM for 4 hours. At the end of incubation, the culture media were collected and directly analyzed for OT concentration by radioimmunoassay (RIA) . Culture medium not exposed to cells served as a control. Antibody, specific for OT nonapeptide (a gift from Dr. M. Morris, Wright State University, Dayton, OH), and synthetic OT standards (Peninsula Laboratories) were used to measure OT concentration.
3 r& B( v$ n& e5 p
Reverse Transcription Polymerase Chain Reaction: I/ `2 I  v: h( T  r/ a

Total RNA was extracted from cells with TRIzol reagent (Invitrogen Life Technologies). For OT transcript analysis, poly(A)  mRNA was affinity purified on Oligotex mRNA columns (Qiagen, Ontario, Canada, http://www.quiagen.com), and mRNAs for endothelial NOS (eNOS), GATA4 and atrial natriuretic peptide (ANP) were analyzed in DNase-treated samples of total RNA (Turbo DNase-free, Ambion Inc., Austin, TX, http://www.ambion.com). The samples were then reverse transcribed to cDNA, amplified by polymerase chain reaction (PCR; Robocycler Gradient 40 Thermocycler, Stratagene, La Jolla, CA, http://www.stratagene.com), and resolved on agarose gel. Bands stained by ethidium bromide were analyzed with the Storm 840 imaging system and ImageQuant software (Version 4.2, Molecular Dynamics, Inc., Sunnyvale, CA, http://www.ump.com/mdynamic.html). To validate this reverse transcriptase (RT)-PCR assay as a tool for the semiquantitative measurement of mRNA, dose-response curves were established for different amounts of total RNA extracted from P19EC cells, and the samples were quantified in the linear phase of PCR amplification. These data were normalized to the corresponding values of 18S RNA PCR products serving as the internal controls (Ambion). The PCR primers and conditions are shown in supplemental Table 1. Sequences of mouse ANP genes have been described  OTA  was a sense-strand primer corresponding to a sequence in exon A, starting three base pairs downstream of the initiation codon. OTB  and OTB¨C were sense- and antisense-strand primers located at the 5' and 3' ends of exon B, respectively. OTC¨C was an antisense primer complementary to a sequence in exon C, terminating at the stop codon. 18 S (kit from Ambion, Canada) gene expression was an internal standard measure in this experiment.4 K3 E: e* K, E5 H3 C1 ~- d" t
, c  N# F# @9 u/ q- f* O" I5 ?0 D
Table 1. Primers
0 P! c3 f+ }1 s
Real-Time Quantitative PCR

Real-time PCR was performed according to standard protocols on a MyIQ Real-Time PCR detection system (Bio-Rad). Briefly, 1 µg of total RNA was DNase-treated and transcribed to cDNA. For amplification, 2 µl of diluted cDNA was added to a 20-µl reaction mixture containing 1x iQ SYBR Green Supermix (Bio-Rad) and 200 nM forward and reverse primers. The thermal cycling program was 95¡ãC for 2 minutes, followed by 40 cycles of 95¡ãC for 30 seconds, 60¡ãC for 30 seconds, and 72¡ãC for 30 seconds. The sequences of primers purchased from Invitrogen Life Technologies are shown in supplemental Table 1. All reactions were run in triplicate. As negative controls, PCR was performed on water and on RNA without reverse transcription. The cycle number at which the reaction crossed an arbitrarily placed threshold (CT) was determined for each gene. The relative amount of mRNA levels was quantified by 2 ¨C CT. Relative gene expression was normalized to glyseraldehyde-3-phosphate dehydrogenase (GAPDH) expression.% o/ j$ t  e' o4 u, m
' [7 c3 }; b: O4 ~4 S& {$ S9 J; g
Statistical Analysis4 n1 e. N0 l% e% T7 s
/ d) e+ v  b8 [6 `
Values are expressed as mean ¡À SEM. Multiple comparisons were made by analysis of variance, followed by Dunnett's modified t test or, in the case of comparison with only one group, by a two-tailed version of Student's t test.

RESULTS
5 Q! s; E  W0 l6 [' e, m
L-NAME Inhibits the Appearance of Beating Colonies in OT-Induced P19EC Cells

Treatment of P19EC cell aggregates with 10¨C7 M OT induced beating cell colonies earlier and in higher numbers than treatment with 0.5% DMSO (Fig. 1A) by day 14 (20 ¡À 0.3 vs. 14 ¡À 0.7 positive wells, respectively; p
, ~2 }5 K. y; o0 K3 k# E
Figure 1. L-NAME inhibits OT- and DMSO-induced generation of beating cells. (A): Time-course of appearance of beating cell colonies upon treatment with different agents. Cell aggregates, NI or induced for 4 days with the indicated agent(s), were evenly distributed in wells of 24-well tissue culture plates. Then, each plate was examined at 2-day intervals for the number of wells containing beating cell colonies. The results (mean ¡À SEM) are from three independent differentiation experiments. Morphology of day 14 contracting cell colonies in the NI condition (B), or following induction with OT (C), OT  L-NAME (D) L-NAME (E), DMSO (F), or DMSO  L-NAME (G). Dotted lines encircle contracting cell colonies. Video images of contracting cell colonies are available for (E¨CG). * p
1 M& ~+ q, M3 r: @" H# F
Beating colonies had a different morphology after the various treatments. As seen in Figures 1C and 1F, OT and DMSO induced spherical beating cell colonies, displaying robust, synchronized contractions. Such colonies were absent in noninduced cultures (Fig. 1B) and in cultures treated with OT  L-NAME (Fig. 1D). Beating cell colonies in DMSO  L-NAME treated cultures sometimes displayed large clusters of frequently beating cells (Fig. 1G), indicating that this treatment still promoted the CM phenotype. In contrast, a few very small, round clusters of weakly beating cells were seen in L-NAME treated cultures (Fig. 1E).1 E$ J2 S5 H8 g2 c# @1 G
  R9 O2 D& [4 R
Changes of Gene Expression During OT-Mediated Cell Differentiation

Changes in GATA4 expression were an early indicator of OT-mediated cell differentiation as assessed by RT-PCR (Fig. 2). The results in Figure 2A demonstrate that, in P19EC cells exposed to OT, GATA4 mRNA was already increased in aggregates on the 4th day of differentiation (fivefold increment compared to day 1). A further elevation of GATA4 mRNA was seen on day 6 (10-fold), when the first beating cell colonies were detected. Maximally increased GATA4 mRNA (15-fold) was evident on days 10 and 14. Unexpectedly, in cells exposed to OT  L-NAME or L-NAME alone, GATA4 mRNA expression was also detected, but at a relatively lower level. In L-NAME-treated cells, a sixfold GATA mRNA increment was noted on days 10 and 14.

Figure 2. Time-course polymerase chain reaction (PCR) analysis of gene expression during cardiomyocyte (CM) differentiation of P19 cells induced by OT, OT  L-NAME or L-NAME alone. Reverse transcriptase (RT)-PCR for GATA4 mRNA (A), endothelial nitric oxide synthase (eNOS) mRNA (B), and ANP (C) mRNA expression relative to 18S RNA. The results (mean ¡À SEM) are from three independent experiments performed in duplicate. *p

As shown in Figure 2B, eNOS mRNA was barely detectable in cell aggregates (days 1 and 4) induced by OT in the presence or absence of L-NAME. Interestingly, in OT-induced cultures, the first appearance of beating CM (day 6) was associated with high eNOS mRNA expression (30 times greater than in cultures on day 1), which remained static through days 10¨C14. At the stage of cardiomyoblast generation on day 4 and with the onset of beating colonies on day 6, the eNOS mRNA level was very low in cells exposed to OT  L-NAME or L-NAME alone. However, after CM formation on days 10 and 14, eNOS expression was significantly augmented in OT  L-NAME induced cells and, to a lesser extent, in cells exposed to L-NAME alone; these augmentations were significantly smaller than in OT-treated cells.
; ?5 R' O; B: ], R  W: H1 A
ANP mRNA increased progressively until day 10 in cells exposed to OT (Fig. 2C). In contrast to effective beating cell inhibition, L-NAME had a less important impact on OT-induced ANP mRNA upregulation (respectively, 20% and 30% reductions at days 10 and 14). Interestingly, a high ANP mRNA level was found in L-NAME treated cultures displaying a few foci of beating cells, suggesting some cardiomyogenic process in NO-deficient cells." i1 a. e( Q" @8 r# z/ D' K
5 N6 T' z9 {7 m' d7 G2 r2 f( V
The expression of other cardiac-specific genes was assessed in developing P19EC cells by real-time PCR. As shown in Figure 2, at a time corresponding to the cardiomyoblast stage (day 4), early CM (day 6), and beating CM (day 10), OT-treated cells expressed the cardiac transcription factors Nkx2.5 (Fig. 2D) and MEF2c (Fig. 2E) as well as the cardiac-specific -MHC (Fig. 2F). At all such stages of cell differentiation, L-NAME largely reduced expression of these genes. Further analysis indicated that P19 cells responded to OT induction by expressing myogenin, the transcription factor participating in the generation of skeletal muscle in P19EC cells . This response was also attenuated in the presence of L-NAME (Fig. 2G).

The observation that, in response to OT treatment, P19EC cells generate both CM and skeletal muscle markers suggests multiple myogenic actions of the hormone. Therefore, we performed a series of experiments to visualize the population of P19 cells expressing CM markers upon stimulation with OT. For this purpose, we utilized P19Cl6-GFP cells induced with OT, L-NAME alone, and OT and L-NAME in combination. In cell cultures collected at day 14 of differentiation and displaying comparable and complete confluence (Fig. 3F), we analyzed the expression of sarcomeric (cardiac and skeletal) -actinin with the antibody complex emitting red fluorescence (Alexa Fluor 568). Brilliant GFP-borne green fluorescence indicated the presence of MLC-2v ventricular marker. Analysis of fluorescence under confocal microscopy suggested colocalization of -actinin and GFP fluorescence in 3.5% ¡À 0.4% of culture areas in OT-induced cell populations, but in only 0.6% ¡À 0.3% and 0.5% ¡À 0.2% of culture areas upon OT  L-NAME and L-NAME treatments, respectively. The cultures stimulated with OT and OT  L-NAME generated similar fractions of cells expressing only -actinin (3.5% ¡À 0.5% and 3.2% ¡À 0.7% respectively). This fraction of cells was lower upon stimulation with L-NAME (1.7% ¡À 0.2%), and possibly contained some skeletal muscle cells (elongated shape) and/or nonventricular CM, indicated by round and triangular cell morphology. Noticeably, almost all GFP-positive cells displayed this latter shape and were induced by OT in a 12.3% ¡À 1.3% proportion. This proportion was reduced to 4.8% ¡À 0.8% by treatment with OT  L-NAME and was 6.7% ¡À 0.3% in cultures induced with L-NAME alone (both p
9 S, a) C" D8 }* Q& w. Y
Figure 3. Representative confocal microscopy analysis of green fluorescence-emitting P19Cl6-GFP cells stained for -actinin with antibodies labeled with Alexa Fluor 568. The cells were induced into differentiation for 4 days with (B) OT, (C) OT  L-NAME, and (D)L-NAME and (A) NI, then collected at differentiation day 14 (bar = 100 µm). (E) The histobars quantitatively illustrate the percentage of image areas emitting only red fluorescence (see arrows B1, -actinin), only GFP fluorescence (B2) and areas of colocalized red and green fluorescence (B3). Calculations were performed using the Image J program and intensity correlation analysis software. In all cases, fluorescence was evaluated on fields exhibiting similar high cell densities (F). The results are presented as mean ¡À SEM of three independent experiments. The symbols * and & respectively indicate differences versus corresponding NI cultures and cultures induced with OT (p 9 k# C2 T, D2 a

Expression of SSEA-1 and Contractile Proteins- ^: p( v& Q7 ^, s0 v# I

In P19EC cells at an advanced stage of differentiation (day 14), we analyzed SSEA-1, the marker of the undifferentiated state. Positive fluorescence staining with monoclonal antibody against SSEA-1 has been demonstrated in noninduced cells (Fig. 4D). A smaller fraction of cells positive for SSEA-1 after induction by OT and/or L-NAME (Fig. 4A¨C4C) indicated that a differentiation process was initiated in these cultures. Secondly, we investigated whether the cells acquire two markers of the advanced contractile apparatus in CM, namely sarcomeric MHC and DHPR . As expected, noninduced cells were negative for DHPR and MHC (Fig. 4H, Fig. 4L), displaying only basal fluorescence. The two contractile proteins were clearly detected in OT-treated cells (Fig. 4E, 4I), whereas OT  L-NAME and L-NAME treated cells exhibited only the low fluorescence of noninduced cells in the overall cultures (Fig. 4F, 4G, 4J, 4K). It is possible that, in addition to CM, some cells in OT- and/or L-NAME-treated cultures differentiated to other, nonmuscular cell phenotypes./ u$ o. Q" Y  v7 ?/ [

Figure 4. Staining of P19 cells for the embryonic cell marker anti-SSEA-1 (A¨CD), DHPR (E¨CH), and sarcomeric MHC (I¨CL) on day 14 of cardiomyogenic differentiation. Cells aggregated for 4 days in 6-cm diameter bacteriological Petri dishes in the absence (NI) or presence of oxytocin (OT), OT  L-NAME, or L-NAME were transferred into 10-cm diameter tissue culture dishes (one per Petri dish) and cultured until day 14. Cells attached to the dishes were immunostained, and representative results are shown (bar = 50 µm). In contrast to OT treatment, only basal fluorescence was observed for MHC and DHPR signals in NI cultures and cultures induced with OT  L-NAME or L-NAME. (M): Calculated fluorescent areas (mean ¡À SEM) from five representative fluoromicrographs for each treatment. * p ) c! z) d- o# ]! V9 V

Endogenous OT Expression in P19EC Cells) m( d" A7 r& M5 ?3 x5 V6 U" i( t
0 G5 D3 f! k0 o% ?
The ability of P19 cells to undergo myogenesis depends in part on unknown factors in serum . It is possible that differentiation depends on endogenous OT expression in P19 cells. Therefore, using RIA, we investigated whether P19EC differentiation is associated with OT production. On day 14 of OT-stimulated differentiation, OT was measured 4 hours after the addition of fresh medium. We detected 6.2 ¡À 0.2 pg of OT per milligram of protein in cell extracts and 14.2 ¡À 0.8 pg/ml in culture medium. Less OT was found in DMSO-stimulated P19EC cells: 3.7 ¡À 0.1 pg/mg of protein in cell extracts and 7.0 ¡À 0.1 pg/ml in medium. Because the contribution of serum-containing culture medium was only 1.1 ¡À 0.04 pg/ml, these results suggest that differentiated P19EC cells produce OT. The presence of OT transcript in OT-stimulated P19EC cells at a period preceding the appearance of beating colonies (day 6 of the differentiation protocol; Fig. 5) further indicates OT synthesis in these cells. In a series of experiments, RT-PCR was performed with OT gene-specific primers homologous to sequences on three exons of the OT gene, as represented schematically in Figure 5A. This strategy was chosen to reveal any structural differences in endogenous OT expression that might exist between the OT-coding regions in P19EC cells. The results reveal that OT-stimulated P19EC cells expressed all coding regions of the OT transcript. Secondly, we found that OT induction of P19EC stimulated OT mRNA expression in comparison to noninduced cells, but combined treatment with L-NAME abolished OT mRNA elevation (Fig. 5B).

Figure 5. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of OT gene expression in OT-stimulated P19 cells. (A): PCR amplification of products obtained with OT exon-specific primers using reverse-transcribed mRNA from P19 cells induced by OT. A C¨C 414-bp OT cDNA products of PCR from primers located on exons 1 and 3; A B¨C 360-bp OT cDNA products of PCR from primers located on exons 1 and 2; and B C¨C 223-bp OT cDNA products of PCR from primers located on exons 2 and 3. (B): OT induction of P19EC cells increases endogenous OT expression, and this effect is abolished by L-NAME. Experiments were performed on NI and OT-induced P19 cells collected on day 6 of the differentiation protocol. OT expression was analyzed with B C¨C primers and expressed relative to 18 S RNA. Relative to P19EC cells, OT is barely detected in the mouse heart. All results are representative of three independent experiments. * p 1 h/ m4 O" p2 x9 W8 {. f( i4 e
* ]& z; x3 s; `- E+ e4 }0 p( o
SNAP Reverses the Inhibitory Effect of L-NAME on OT-Induced Cardiomyogenesis
' A1 k: g' ^# n/ k1 f
In P19Cl6-GFP cultures, OT (22 wells) induced the generation of beating colonies more efficiently did than SNAP (18 wells), an NO generator (Fig. 6A). The number of wells with OT-induced contracting cells was reduced in the presence of L-NAME (4¨C5 wells) to levels seen in basal, spontaneous cardiomyogenic differentiation (noninduced, 3¨C4 wells). Compared with P19EC, where no beating cell colonies were observed in the absence of inducer (Fig. 1), some, although very rarely, contracting cells were spontaneously generated in P19Cl6-GFP cultures (Fig. 6A). When OT  L-NAME treatment was given in the presence of SNAP, the number of contracting cultures increased from 4¨C5 to 17. Thus, NO supplementation partially reversed the inhibitory effect of L-NAME on OT-induced cardiomyogenesis.

Figure 6. SNAP reverses L-NAME inhibition on OT-induced cardiomyogenesis. (A): Time-course of appearance of beating colonies of P19Cl6-GFP cells NI or induced with OT, L-NAME, SNAP, or their combinations. Induction treatment is described in the Figure 1 legend. The results (mean ¡À SEM) are from three independent cultures. (B¨CG) Representative green fluorescent protein (GFP) fluoromicrographs (day 10, bar = 50 µm) of NI cultures (B), and cultures induced with OT (C), OT  L-NAME (D), OT  L-NAME  SNAP (E), SNAP (F), and OT  SNAP (G). (H:) Calculated fluorescent areas (mean ¡À SEM) from three representative fluoromicrographs for each treatment. * p 3 P4 R/ P( `1 G1 p

Fluorescence microscopy was employed to visualize both MLC-2v-GFP staining intensity and distribution in response to the differentiation inducers. Figures 6B¨C6G present fluoromicrographs taken on day 10 of differentiation. Only basal fluorescence was seen in noninduced cultures (Fig. 6B). OT-treated cultures produced intense GFP fluorescence areas (Fig. 6C). OT induction of MLC-2v-GFP expressing cells was reduced in the presence of L-NAME (Fig. 6D). SNAP partially reversed the inhibitory effect of L-NAME on OT-induced CM differentiation (Fig. 6E). SNAP exhibited large areas of weak fluorescence studded with intensively-emitting spots that enlarged upon combination with OT treatment (Fig. 6F, FG). High fluorescence-emitting areas, such as those seen in OT-differentiated cultures, overlapped the sites of contracting cell colonies (Fig. 6C). In contrast, areas of weak fluorescence were not always associated with beating (e.g., OT  L-NAME; Fig. 6D). These results indicate OT effectiveness in CM differentiation to the ventricular phenotype and NO involvement in this process.% R5 B1 l5 y+ {" |% H; q

The formation of beating cell colonies expressing CM ventricular MLC-2v marker in response to both NO generation and inhibition of the NO pathway (albeit with different efficiencies) pointed to the pleiotropic action of NO in cardiomyogenesis. In this respect, we found that OT as well as L-NAME treatments of P19EC cell monolayers elicited proliferative actions, as assessed by crystal violet staining (1.6- ¡À 0.12-fold and 2.0- ¡À 0.11-fold higher cell numbers respectively vs. noninduced cells; p " f) h6 n" @5 G, e( I1 N! q. _

Inhibition of Inducible NOS and NO-Sensitive Soluble Guanylyl Cyclase Reduces OT-Evoked Cardiomyogenesis  E* |4 F- k4 W7 m
& d% c) i; S3 p# {
To better understand the NO pathway involved in OT-induced cardiomyogenesis, we studied the effect of 1400W, a specific inhibitor of inducible NOS (iNOS) . Figure 7A shows that both inhibitors decreased the number of contracting colonies generated by OT treatment (OT, 16 wells on day 14; OT  1400W, 7 wells; and OT  ODQ, 8 wells) and, in the absence of OT, they stimulated rare and undersized contracting cell clusters as well as MLC-2v mediated fluorescence (Fig. 7B).
; L2 ~5 e2 z5 P; y
Figure 7. Inhibition of inducible nitric-oxide synthase (iNOS) and nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) reduces oxytocin (OT)-induced cardiomyogenesis. (A) Comparison of the time-course generation of contracting colonies in P19Cl6-GFP cells noninduced (NI) or induced with OT, 1400W, ODQ, or their combinations. The ODQ curve is hidden by the OT  ODQ curve. Treatment was performed as described in the Figure 1 legend. The results (mean ¡À SEM) are from three independent experiments. (B) Calculated fluorescent areas (mean ¡À SEM) from 16 representative fluoromicrographs for each treatment. * p ! \4 N( Z4 F+ G2 M1 t3 C

DISCUSSION7 S' ]7 s8 a7 n, n# i5 [

This report follows up studies on the mechanism of OT-induced stem cell differentiation into CM. In previous investigations, we have provided evidence that OTR mRNA expression is enhanced during CM differentiation of pluripotent P19EC cells . Here, we demonstrated that NO contributes to OT-induced CM differentiation through a pathway involving eNOS mRNA upregulation as well as iNOS- and NO-dependent sGC activity. Image analysis of P19Cl6-GFP cells induced by OT revealed the presence of ventricular CM coexpressing sarcomeric -actinin and MLC-2v specific GFP, with some cells exclusively expressing -actinin, most likely skeletal myocytes (SMs). The OT-mediated production of CM and, less evidently, the SMs was reduced by L-NAME supplementation. We found that inhibition of the NO pathways in OT-mediated CM differentiation significantly reduced the generation of functional, contracting CM, indicating that NO is a transducing molecule in the cascade of intracellular events. This was further supported by reversal of the L-NAME effect with the NO donor SNAP.! G  B) K( W5 q, i8 {1 u( u

The present work shows the functional involvement of iNOS/eNOS/sGC in OT-mediated CM differentiation. This was demonstrated by blockage of the NO pathways and by OT stimulation of eNOS mRNA (stimulation inhibited by L-NAME). The iNOS transcript was undetected under our conditions, but iNOS action was indicated by sensitivity to its specific inhibitor, 1400W. NO is a ubiquitous signaling molecule, characterized by high reactivity but with a self-limiting duration of action. NO acts through cGMP-dependent and -independent pathways to regulate gene expression by modulating transcription factors, translation, or the stability of mRNA and proteins .

We found that NO plays an important role in OT-mediated CM maturation of P19EC cells because inhibition of the NO pathway drastically reduced beating cell colonies, Nkx2.5 and MEF2c transcripts, and contractile protein expression, although there was a relatively less pronounced reduction of GATA4 and ANP transcripts. Interestingly, OT exhibited a more efficient cardiomyogenic action than did SNAP, as assessed by the number of beating cell cultures and their size and MLC-2v associated GFP expression. This could be due to fine regulation of NO at the OTR level and/or the contribution of additional NO-independent transduction pathways triggered by OT.
3 n/ i) ?3 Q3 S3 O+ p
The optimal OT concentration for CM induction was in the range of pharmacological concentrations (10¨C7 M). Under these conditions, as with other G protein-coupled receptors, it is likely that OTRs internalize and/or desensitize upon continuous agonist exposure, which was indicated in other models by changes in intracellular calcium mobilization . Therefore, we could speculate that under conditions of CM differentiation of P19EC cells, OTRs undergo a similarly extensive and rapid process of desensitization and internalization after OT exposure, with changes in intracellular calcium and NO mobilization. It is possible that this cycle of events is necessary to trigger OT-mediated cardiomyogenesis. Thus, NO deficits during the OTR desensitization period can provide a part of the signaling required to initiate cardiomyogenesis. Correspondingly, we found that L-NAME alone, as well as 1400W and ODQ, two other inhibitors of NO pathways, initiated some cardiomyogenesis in P19EC cells. L-NAME downregulated the embryonic cell marker SSEA-1, and increased GATA4 and ANP transcripts as well as MLC-2v associated GFP reporter gene. L-NAME, however, induced only a few small, round clusters of weakly beating cell colonies and hardly stimulated the synthesis of MHC and DHPR markers displayed in terminally differentiated P19EC. In P19Cl6-GFP cultures, L-NAME induced some cells expressing -actinin and SMs marker myogenin. However, L-NAME treatment did not influence the OT-mediated production of cells expressing -actinin, while being negative for the ventricular cell marker MLC-2v. These observations indicate that, in P19 cells induced by OT, L-NAME predominantly inhibits the production of ventricular CM and has a minimal effect on simultaneously-generated populations of SMs.* |- R7 N. V$ f' l' [

It is possible that the decrease of NO by L-NAME can remove a repressive barrier on differentiation and/or provide an inductive clue to initiate myogenesis but is insufficient to complete CM differentiation and maturation. Correspondingly, in cancer cells, high NO levels may be cytostatic or cytotoxic, whereas low-level activity can have the opposite effect and promote cell growth . Therefore, the tuning of potentially positive and negative NO actions and the molecular recognition of these balances will be central to understanding the role of NO during CM differentiation.
5 ?; C0 _) \" ]" j; h$ r
The role of NO signaling in ES cell-derived CM remains unsolved. Recent studies of Krumenacker et al. .

Endogenous OT produced in P19EC cells can act in paracrine and autocrine ways to provide the secondary stimulus triggering further steps of CM differentiation. In accordance with this hypothesis, our experiments showed that OT gene expression in P19EC cells was enhanced after OT induction and was lowered in the presence of L-NAME. OT RIA in P19EC cells revealed that, although most of the peptide was targeted for secretion, consistent amounts of OT were also found in cells, raising the additional possibility of an "intracrine" action of the hormone in cells coexpressing OT and its receptor. This OT upregulation occurring early in induced cells (day 6) could provide a means to sustain NO production (for instance, through eNOS upregulation), even after removal of the exogenously supplied hormone. In addition, the stimulatory effect of OT on P19EC cell proliferation could facilitate NO-mediated cell selection and differentiation.
0 J$ r, \3 p- l  f* C& p
It has been shown that, in DMSO-induced P19EC cells, the expression of GATA4 transcript and protein is restricted to cells committed to the cardiac lineage, and GATA4 induction precedes the expression of cardiac marker genes as well as the appearance of beating cells .

In our studies, whereas GATA4 expression was only moderately reduced when L-NAME was administered together with OT, the factors MEF2c and Nkx2.5 were extensively downregulated. This lack of balance of transcription factors can severely impair the cardiomyogenic program, which requires physical interaction and synergistic modulation of target gene expressions . Most likely, free radical generation is not required for OT-induced GATA4 expression, indicating that a finely tuned interplay between OT-dependent and -independent transcription factors may be required for proper cardiomyogenic differentiation.% @6 A% n: y" }& q
$ W7 X+ A9 C( M" Q+ e  l" d+ B
In summary, we found that OT upregulated eNOS expression during differentiation of mouse P19EC cells into CM, and that inhibition of NO signaling during this process reduced CM yield. The NO generated by SNAP treatment, although cardiomyogenic, did not enhance CM differentiation induced by OT. Moreover, beating colonies were larger in OT- than in SNAP-induced cultures. This suggests that additional signaling pathway(s) besides NO are involved in OT-stimulated cardiomyogenesis. Because NO inhibition, in addition, initiates CM differentiation, we postulate that NO influences cardiac differentiation in P19EC cells through a pleiotropic mechanism.
# S9 F  c- n2 Y/ c% L
DISCLOSURES

The authors indicate no potential conflicts of interest.4 i1 [; C$ i8 b* d

ACKNOWLEDGMENTS7 }$ O. w6 L& @
9 v( }$ w' l" a! E8 j/ d
We acknowledge the editorial work of Ovid Da Silva and the secretarial assistance of Antoinette Paolitto. B.D. is the recipient of a studentship from Fonds Qu¨¦b¨¦cois de la Recherche sur la Nature et les Technologies and a studentship from Universit¨¦ du Qu¨¦bec ¨¤ Montr¨¦al. This work was supported by Canadian Institutes of Health Research and Canadian Heart and Stroke Foundation Grants MOP-53217 (J.G. and M.J.), and NET SRD-63193 (J.G., M.J., and J.P.).
      【参考文献】' b+ t  @' }3 B  e% }, Q2 Z6 O
( N" J8 @1 z, g: ^& Z& L& B
! F) {6 t+ F. i5 Y
Wobus AM, Boheler KR. Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol Rev 2005;85:635¨C678.
7 M$ A9 r3 t0 L1 T) g1 L" U
van der Heyden MA, Defize LH. Twenty one years of P19 cells: What an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res 2003;58:292¨C302.* F: s4 O; \0 _7 V

Skerjanc IS. Cardiac and skeletal muscle development in P19 embryonal carcinoma cells. Trends Cardiovasc Med 1999;9:139¨C143.8 Y- Y+ J. G  f  G$ m% c& g
+ @) L- [" D9 {- H2 s2 r
van der Heyden MA, van Kempen MJ, Tsuji Y et al. P19 embryonal carcinoma cells: A suitable model system for cardiac electrophysiological differentiation at the molecular and functional level. Cardiovasc Res 2003;58:410¨C422.
4 _$ u7 T% i1 B9 o
Moore JC, Spijker R, Martens AC et al. A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells. Int J Dev Biol 2004;48:47¨C55.3 I4 U  K! d  E5 V

Habara-Ohkubo A. Differentiation of beating cardiac muscle cells from a derivative of P19 embryonal carcinoma cells. Cell Struct Funct 1996;21:101¨C110.

Gutkowska J, Jankowski M, Lambert C et al. Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart. Proc Natl Acad Sci U S A 1997;94:11704¨C11709.

Jankowski M, Hajjar F, Kawas SA et al. Rat heart: A site of oxytocin production and action. Proc Natl Acad Sci U S A 1998;95:14558¨C14563.: E  [- [7 v( O/ f
; H% a% U& D' G2 C9 l7 l
Paquin J, Danalache BA, Jankowski M et al. Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes. Proc Natl Acad Sci U S A 2002;99:9550¨C9555.

Jankowski M, Danalache B, Wang D et al. Oxytocin in cardiac ontogeny. Proc Natl Acad Sci U S A 2004;101:13074¨C13079.( U/ @# k4 W3 y6 @, d; \, c" Z' F

Matsuura K, Nagai T, Nishigaki N et al. Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes. J Biol Chem 2004;279:11384¨C11391.) ^% [3 g5 h6 M; I

Gimpl G, Fahrenholz F. The oxytocin receptor system: Structure, function, and regulation. Physiol Rev 2001;81:629¨C683.
3 H1 U8 W' G9 f' l4 H
Thibonnier M, Conarty DM, Preston JA et al. Human vascular endothelial cells express oxytocin receptors. Endocrinology 1999;140:1301¨C1309.

Haraldsen L, Soderstrom-Lauritzsen V, Nilsson GE. Oxytocin stimulates cerebral blood flow in rainbow trout (Oncorhynchus mykiss) through a nitric oxide dependent mechanism. Brain Res 2002;929:10¨C14.) c, T& L2 L1 k( P$ l
0 z. I+ C' ]0 r+ ^
Chiodera P, Volpi R, Manfredi G et al. Effect of oxytocin on nitric oxide activity controlling gonadotropin secretion in humans. Eur J Clin Invest 2003;33:402¨C405.
8 f  u; O  }. z" F
Selvage DJ, Johnston CA. Interaction between norepinephrine, oxytocin, and nitric oxide in the stimulation of gonadotropin-releasing hormone release from proestrous rat basal hypothalamus explants. J Neuroendocrinol 2004;16:819¨C824.' K6 K  |$ ]8 |* O' M

Mukaddam-Daher S, Yin YL, Roy J et al. Negative inotropic and chronotropic effects of oxytocin. Hypertension 2001;38:292¨C296.
9 m9 _9 P: Y/ H2 e$ E
Jankowski M, Wang D, Mukaddam-Daher S et al. Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle. J Endocrinol 2005;184:209¨C217.
: D+ u- K4 ]. |5 ?; Q1 J# @
Bloch W, Fleischmann BK, Lorke DE et al. Nitric oxide synthase expression and role during cardiomyogenesis. Cardiovasc Res 1999;43:675¨C684.6 n& p0 @" ~9 _! ?% K) R' z9 v

Kanno S, Kim PK, Sallam K et al. Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells. Proc Natl Acad Sci U S A 2004;101:12277¨C12281.% u1 F% P' m: G
1 X9 o. b5 Y! z9 M, z, ^
Laplante I, Paquin J, Beliveau R. RhoB expression is induced after the transient upregulation of RhoA and Cdc42 during neuronal differentiation and influenced by culture substratum and microtubule integrity. Dev Brain Res 2001;129:157¨C168.

Nemer G, Nemer M. Transcriptional activation of BMP-4 and regulation of mammalian organogenesis by GATA4 and -6. Dev Biol 2003;254:131¨C148.

Lefebvre DL, Giaid A, Bennett H et al. Oxytocin gene expression in rat uterus. Science 1992;256:1553¨C1555.8 c+ c; S" \- K# m
$ v' K6 {3 l4 k" j6 C% ]6 ?8 m
Ridgeway AG, Wilton S, Skerjanc IS. Myocyte enhancer factor 2C and myogenin up-regulate each other's expression and induce the development of skeletal muscle in P19 cells. J Biol Chem 2000;275:41¨C46.

Wilton S, Skerjanc IS. Factors in serum regulate muscle development in P19 cells. In Vitro Cell Dev Biol Anim 1999;35:175¨C177.
9 Y+ `; @: r: Q6 C& m! P: H
Jeng YJ, Soloff SL, Anderson GD, Soloff MS. Regulation of oxytocin receptor expression in cultured human myometrial cells by fetal bovine serum and lysophospholipids. Endocrinology 2003;144:61¨C68.8 M' i3 y3 v0 e) s4 m

Garvey EP, Oplinger JA, Furfine ES et al. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem 1997;272:4959¨C4963.
* ^2 s9 m2 l' e) n+ |
Garthwaite J, Southam E, Boulton CL et al. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-1 l; q. t8 a4 X: R7 K3 H& y

Bogdan C. Nitric oxide and the regulation of gene expression. Trends Cell Biol 2001;11:66¨C75.

Braam B, de Roos R, Dijk A et al. Nitric oxide donor induces temporal and dose-dependent reduction of gene expression in human endothelial cells. Am J Physiol 2004;287:H1977¨CH1986.

Suzuki YJ, Nagase H, Day RM et al. GATA4 regulation of myocardial survival in the preconditioned heart. J Mol Cell Cardiol 2004;37:1195¨C1203.5 c* x3 v5 c* m

Smith MP, Ayad VJ, Mundell SJ et al. Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and Clathrin mutants in HEK-293 cells. Mol Endocrinol 2006;20:379¨C388.
  W& }! @  C. W6 U2 O  X4 d5 ?
Phaneuf S, Asboth G, Carrasco MP et al. The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA. J Endocrinol 1997;154:7¨C18.4 k. \# I  _% i4 H! A# B0 M

Phaneuf S, Asboth G, Carrasco MP et al. Desensitization of oxytocin receptors in human myometrium. Hum Reprod Update 1998;4:625¨C633.& G- v& ~# }  \9 G& s0 O, R

Xu W, Liu LZ, Loizidou M et al. The role of nitric oxide in cancer. Cell Res 2002;12:311¨C320.6 x+ A& K7 W: [! g& Z/ b
; _6 l2 D9 j1 k$ V2 ?
Berg RW, McBurney MW. Cell density and cell cycle effects on retinoic acid-induced carcinoma cell differentiation. Dev Biol 1990;138:123¨C135.9 @* R- s. z/ w- x- u( G3 J

Krumenacker JS, Katsuki S, Kots A et al. Differential expression of genes involved in cGMP-dependent nitric oxide signaling in murine embryonic stem (ES) cells and ES cell-derived cardiomyocytes. Nitric Oxide 2006;14:1¨C11.0 u+ @6 t; X* a$ z; P5 k

Bu S, Xia G, Tao Y, Lei L, Zhou B. Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro. Mol Cell Endocrinol 2003;207:21¨C30.

Thaler CD, Epel D. Nitric oxide in oocyte maturation, ovulation, fertilization, cleavage and implantation: A little dab'll do ya. Curr Pharm Des 2003;9:399¨C409.# W! z9 t! V* ~7 n, c) Z
/ G& v& m0 \( w4 [
Sato N, Meijer L, Skaltsounis P et al. Maintenance of pluripotency in human and embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55¨C63.

Ahumada A, Slusarski DC, Liu X et al. Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP. Science 2002;298:2006¨C2010.

Gr¨¦pin C, Robitaille L, Antakly T et al. Inhibition of transcription factor GATA4 expression blocks in vitro cardiac muscle differentiation. Mol Cell Biol 1995;15:4095¨C4102.
" y( V' W) }. P$ a& a( |
Liu H, Harris TM, Kim HH et al. Cardiac myocyte differentiation: the Nkx2.5 and Cripto target genes in P19 clone 6 cells. Funct Integr Genomics 2005;5:218¨C239.

Weiss A, Leinwand LA. The mammalian myosin heavy chain gene family. Annu Rev Cell Dev Biol 1996;12:417¨C439./ ]+ h, d  |4 v$ x2 X! Y

Morkin E. Control of cardiac myosin heavy chain gene expression. Microsc Res Tech 2000;50:522¨C531.5 j8 J7 j" F" b6 @) s  c1 y" U4 {
6 c: `- r" k7 T' X; @6 d
Wobus AM, Kleppisch T, Maltsev V et al. Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2  channels. In Vitro Cell Dev Biol Anim 1994;30A:425¨C434.8 X' B! f7 y/ O! B+ u0 H
+ E' e+ f% g& h9 }! k( Q3 n7 ^0 ?
Zheng Z, Wang ZM, Delbono O. Charge movement and transcription regulation of L-type calcium channel alpha (1S) in skeletal muscle cells. J Physiol 2002;540:397¨C409.( i3 _) C( w+ o
+ [! z9 H4 x6 @; b/ i
Small EM, Krieg PA. Transgenic analysis of the atrial natriuretic factor (ANF) promoter: Nkx2¨C5 and GATA-4 binding sites are required for atrial specific expression of ANF. Dev Biol 2003;261:116¨C131.

Uchida S, Fuke S, Tsukahara T. Upregulations of Gata4 and oxytocin receptor are important in cardiomyocyte differentiation processes of P19CL6 cells. J Cell Biochem 9 7, 2006; .4 q" c- k. t' s( }
. Y- h8 ~5 f. O) Y7 k' F8 }- f! @1 ]8 u
Chun YS, Hyun IY, Kwak YG et al. Hypoxicactivation of the atrial natriuretic peptide gene promoter trough direct and indirect actions of hypoxia-inductible factor-1. Biochem J 2003;370:149¨C157.& v4 ~" _2 O* b0 t

Hagen T, Taylor CT, Lam F et al. Redistribution of intracellular oxygen in hypoxia by nitric oxide: Effect of HIF1alpha. Science 2003;302:1975¨C1978.7 `6 C6 U8 J" T* E

Schmelter M, Ateghang B, Helmig S et al. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-indued cardiovascular differentiation. FASEB J 2006;20:1182¨C1184.

收藏0 分享0 顶0 踩0